The overall goal of this proposal is to determine the potential physiological roles of the various NHE isoforms in the intestine. All four of the known NHEs are expressed in the gut, although their roles are not well defined. However, based on our studies of their regional and cellular distributions and their characterization in transfected, NHE-deficient mutant PS120 fibroblasts, we propose that NHE-2 and NHE-3 are involved in vectorial transport and NHE-1 and NHE-4 are activated by acid and luminal osmotic loads. Four cell models will be utilized. 1) Clonal NHE transfectants of PS120 fibroblasts, a cell line that is lacking any form of the exchanger. 2) NHE transfected and non-transfected colon carcinoma cell line Caco-2 as an epithelial model. 3) The non-transformed porcine intestinal epithelial cell line, IPEC, which exhibits vectorial Na transport in response to corticosteroids, as a physiological intestinal cell model. 4) The rat intestine as an intact animal model. The first aim is to confirm the locations of NHE-2 and NHE-3 to the brush border membrane (BBM) and to characterize their regulation and role in vectorial Na transport. The kinetic properties, amiloride sensitivities and regulation will be compared first in PS120 transfectants and then any epithelial-specific features characterized in the epithelial cell lines. It will be determined if protein phosphorylation events are involved in short-term regulation by second messengers. The relevant sites of phosphorylation will be determined by two-dimensional peptide mapping and microsequencing. Next the effects of site-directed mutagenesis, deletions and chimeric recombination of these domains on function and regulation will be determined. Comparison of the transfected fibroblasts and epithelial cells should reveal important differences associated with the regulation and vectorial functioning of these transporters. Long-term regulation by corticosteroids will also be examined. It will be determined if the modulation is at the transcriptional or post-transcriptional level. In the second aim, the hypotheses that NHE-4 and NHE-1 are located on the basolateral membrane (BLM) and play a role in volume and pH regulation to various luminal perturbations will be tested. To confirm preliminary findings that NHE-4 is only activated in response to hyperosmolar stress, the kinetics and regulation of this exchanger will be examined in clonal transfectants of PS120. Once the characteristics have been established, NHE-4 will be studied in the more "physiological" milieu of the epithelial cells. The regulation, physiological role, and phosphorylation-dependence on regulation will be studied. Detailed analyses of the functional properties and regulation of the various isoforms will improve our understanding of the role of the multiple NHE exchangers in normal physiology and provide insights as to their aberrant regulation in potentially life threatening diseases.